Early detection of drug-resistant mycobacterium tuberculosis

ABSTRACT

The present invention relates to oligonucleotides, methods, and kits for detecting an antibiotic-resistant subpopulation within a heteroresistant population of  Mycobacterium tuberculosis  in a sample. An amplicon of a target locus is obtained from the sample. The target locus comprises a region of interest which comprises one or more minor variants associated with the antibiotic resistance. The target locus is selected from the group consisting of: Rv0678, pepQ, atpE, ddn, fbiA, fbiB, fbiC, fgd, fgd1, and fgd2. The region of interest is interrogated to detect the one or more minor variants and thus, the antibiotic-resistant subpopulation of  M. tuberculosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Application No. 63/040,361, filed Jun. 17, 2020, the contents of which are hereby incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII-formatted sequence listing with a file named “91482-175PAT1_Sequence_Listing.txt” created on Jun. 16, 2021, and having a size of 28,419 bytes, is filed concurrently with the specification. The sequence listing contained in this ASCII-formatted document is part of the specification and is herein incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with governmental support under award number R01AI131939 awarded by the National Institutes of Health (NIH). The United States government has certain rights in the invention.

TECHNICAL FIELD

The invention relates to methods, primers, and kits for the early detection of drug-resistant Mycobacterium tuberculosis subpopulation in a sample.

BACKGROUND OF THE INVENTION

Multi drug-resistant (MDR) and extensively drug-resistant Mycobacterium tuberculosis are increasing worldwide. M. tuberculosis does not naturally contain plasmids, and almost all cases of clinical drug-resistance are caused by single-nucleotide polymorphisms (SNPs) or small insertions/deletions in relevant genes. Heteroresistance, which the simultaneous occurrence of drug-resistant subpopulations in an otherwise drug-susceptible bacterial population in a patient, has created uncertainty in the treatment and diagnosis of tuberculosis. Heteroresistance is thought to be an important driver of multi-drug resistance in M. tuberculosis.

Tuberculosis heteroresistance occurs in 9-30% of M. tuberculosis populations studied, and it has been identified in M. tuberculosis populations with phenotypic resistance to first line-drugs (isoniazid, INH; rifampin, RIF; ethionamide, ETA; and streptomycin, S) and second-line fluoroquinolones (ofloxacin, OFX) and injectables (amikacin, AMK). It is highly likely that drug-resistant organisms are present in most tuberculosis lesions, even as very minor population components, given the high bacilli loads that are typically found in patients.

Accordingly, there is a need for oligonucleotides, methods, and kits useful for rapid, molecular, and phenotypic susceptibility assays to identify and/or quantify M. tuberculosis susceptible or resistant to a drug. In particular, there is a need for compositions and methods useful for early detection and/or quantification minor resistance variant subpopulations in clinical samples to allow for effective treatment of tuberculosis.

SUMMARY

The present invention is directed to a method of detecting and/or quantifying a drug-resistant subpopulation of Mycobacterium tuberculosis in a sample, comprising: obtaining an amplicon from the sample, wherein the amplicon comprises a region of interest in Rv0678, pepQ, atpE, ddn, fbiA, fbiB, fbiC, fgd, fgd1, fgd2, or a combination thereof, and the region of interest comprises a polymorphism associated with the drug-resistant subpopulation; sequencing the amplicon; and detecting and/or quantifying a minor variant of the polymorphism in the amplicon, wherein the presence of the minor variant indicates the presence of the drug-resistant subpopulation.

In some aspects, the drug-resistant subpopulation of Mycobacterium tuberculosis is resistant to a bedaquiline-related quinolone derivative, a nitroimidazole antibiotic, or both.

In other aspects, obtaining the amplicon comprises generating the amplicon using at least one primer comprising a sequence with at least 85% identity to a sequence set forth in SEQ ID NOs: 1-168 or a complement thereof.

In one aspect, the minor variant is selected from the group consisting of: a single nucleotide polymorphism (SNP), an insertion, a deletion, and combinations thereof.

In another aspect, the minor variant comprises an insertion or deletion in Rv0678 at position 132, 136, 137, 138, 139, 192, 193, or a combination thereof. In a certain aspect, the minor variant comprises an insertion of G or GA at position 138, an insertion of T at position 139, an insertion of G at position 192, a deletion of G at position 193, or a combination thereof.

In other aspects, the minor variant comprises a SNP in atpE at position 201, 223, or a combination thereof. In one aspect, the minor variant comprises a SNP at position 201 where C is replaced with A or G, a SNP at position 223 where G is replaced with C or T, or a combination thereof.

In some aspects, the region of interest comprises a polymorphism in Rv0678 associated with the bedaquiline-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 20-30 and a complement thereof.

In other aspects, the region of interest comprises a polymorphism in pepQ associated with the bedaquiline-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 1-19 and a complement thereof.

In other aspects, the region of interest comprises a polymorphism in atpE associated with the bedaquiline-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 31-34 and a complement thereof.

In other aspects, the region of interest comprises a polymorphism in ddn associated with the nitroimidazole-resistant subpopulation, and at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 35-36, 133-138, and a complement thereof.

In other aspects, the region of interest comprises a polymorphism in fbiA associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 37-38, 59-60, 77-90, and a complement thereof.

In other aspects, the region of interest comprises a polymorphism in fbiB associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 39-42, 55-58, 73-76, 91-106, and a complement thereof.

In other aspects, the region of interest comprises a polymorphism in fbiC associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 49-54, 67-72, 139-168, and a complement thereof.

In other aspects, the region of interest comprises a polymorphism in fgd associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 43-48, 61-66, and a complement thereof.

In other aspects, the region of interest comprises a polymorphism in fgd1 associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 107-118 and a complement thereof.

In other aspects, the region of interest comprises a polymorphism in fgd2 associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 119-132 and a complement thereof.

In certain aspects, the method further comprises aligning the sequencing data using an alignment algorithm and interrogating the aligned sequencing data to detect and/or quantify the minor variant of the polymorphism.

In one aspect, the steps of sequencing the amplicon and detecting and/or quantifying a minor variant of the polymorphism in the amplicon comprise sequencing two complementary strands of each amplicon to obtain independent sequencing reads of the minor variant and calling the minor variant only when the independent sequencing reads of the minor variant are identical.

In another aspect, the sample is selected from the group consisting of: sputum, pleural fluid, blood, saliva, and combinations thereof from a subject.

In yet other aspects, the method further comprises predicting phenotypic M. tuberculosis resistance to bedaquiline, nitroimidazole, or both, based on a micro-heteroresistance threshold. In one aspect, the micro-heteroresistance threshold is about 5.0%.

In some aspects, the method further comprises administering to the subject a therapeutic agent customized based on the drug resistance of the M. tuberculosis subpopulation in the sample. In one aspect, the therapeutic agent is selected from the group consisting of: an antibiotic, PA-824, OPC-67683, SQ109, TMC207, NAS-21, NAS-91, and combinations thereof.

In some aspects, the present invention relates to a primer for detecting and/or quantifying a drug-resistant subpopulation of Mycobacterium tuberculosis in a sample, the primer comprising a sequence with at least 85% identity to a sequence set forth in SEQ ID NOs: 1-168 or a complement thereof and a label or a modified nucleotide; wherein the primer is between 10 to 70 nucleotides in length. In one aspect, the sequence of the primer consists of a sequence set forth in SEQ ID NOs: 1-168 or a complement thereof; and a label or a modified nucleotide.

In other aspects, the present invention relates to a kit for detecting and/or quantifying a drug-resistant subpopulation of Mycobacterium tuberculosis in a sample, comprising a primer of claim 26 and reagents for amplification of a genomic sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 depict the chronology of the diagnosis and treatment of a case of tuberculosis.

DETAILED DESCRIPTION

Aspects and applications of the invention presented herein are described below in the detailed description of the invention. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts. Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes.

In the following description, and for the purposes of explanation, numerous specific details are set forth to provide a thorough understanding of the various aspects of the invention. It will be understood, however, by those skilled in the relevant arts, that the present invention may be practiced without these specific details. The full scope of the inventions is not limited to the specific examples that are described below. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

It is understood that the disclosed invention is not limited to the particular methodology, protocols, and materials described as these can vary. It is also understood that the terminology used herein is for the purposes of describing embodiments only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

As used herein, the verb “comprise” as is used in this description and the claims and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Also, reference to an element by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements are present unless the context clearly requires that there is one and only one of the elements. The indefinite article “a” or “an” thus usually means “at least one”.

As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

As used herein, the term “detecting” refers to determining the presence or absence of.

As used herein, the term “quantifying” “quantitating” refers to determining the specific amount or ratio of.

As used herein, the term “sample” refers to a sample of biological tissue or fluid that comprises nucleic acids. Such samples include, but are not limited to, tissue isolated from animals. Samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood, plasma, serum, sputum, saliva, stool, tears, mucus, hair, and skin. A sample may be provided by removing a sample of cells from an animal but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Such samples may also include all clinical samples, including for example, cells, tissues, and bodily fluids, such as: blood; derivatives and fractions of blood, such as serum; extracted galls; biopsied or surgically removed tissue, including tissues that are, for example, unfixed, frozen, fixed in formalin and/or embedded in paraffin; tears; milk; skin scrapes; surface washings; urine; sputum; cerebrospinal fluid; prostate fluid; pus; or bone marrow aspirates.

As used herein, the term “antibiotic” refers to a drug (medicine) that inhibits the growth of or destroys Mycobacterium. tuberculosis.

A “modified nucleotide” in the context of an oligonucleotide refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced by a different nucleotide that provides a desired property to the oligonucleotide. Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, e.g., a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, a C5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, a C7-propynyl-dA, a C7-propynyl-dG, a C5-propargylamino-dC, a C5-propargylamino-dU, a C7-propargylamino-dA, a C7-propargylamino-dG, a 7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, a nitro pyrrole, a nitro indole, 2′-O-methyl Ribo-U, 2′-O-methyl Ribo-C, an N4-ethyl-dC, an N6-methyl-dA, and the like. Many other modified nucleotides that can be substituted in the oligonucleotides are referred to herein or are otherwise known in the art. In certain embodiments, modified nucleotide substitutions modify melting temperatures (Tm) of the oligonucleotides relative to the melting temperatures of corresponding unmodified oligonucleotides. To further illustrate, certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation or the like), increase the yield of an intended target amplicon, and/or the like in some embodiments. Examples of these types of nucleic acid modifications are described in, e.g., U.S. Pat. No. 6,001,611, which is incorporated herein by reference.

The term “complement thereof” refers to nucleic acid that is both the same length as, and exactly complementary to, a given nucleic acid.

The terms “identical” or percent “identity” in the context of two or more nucleic acid sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, e.g., as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection. Exemplary algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST programs, which are described in, e.g., Altschul et al. (1990) “Basic local alignment search tool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification of protein coding regions by database similarity search” Nature Genet. 3:266-272, Madden et al. (1996) “Applications of network BLAST server” Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs” Nucleic Acids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation” Genome Res. 7:649-656, which are each incorporated herein by reference.

As used herein, the term “amplicon” refers to a piece of DNA or RNA that is the source and/or product of amplification or replication events.

As used herein, the term “target locus” refers to a fixed position on a M. tuberculosis chromosome, such as the position of a gene or a marker.

As used herein, the term “drug resistance” or “antibiotic resistance” refers to the ability of M. tuberculosis to resist the effects of an antibiotic.

As used herein, the term “region of interest” refers to contiguous or noncontiguous DNA sequence of the target locus identified for a particular purpose. In some aspects, the region of interest refers to a contiguous region of at least 2 nucleotides and less than 500 nucleotides. In other aspects, the region of interest refers to a contiguous region of at least 2 nucleotides and less than 400 nucleotides, at least 2 nucleotides and less than 300 nucleotides, at least 2 nucleotides and less than 200 nucleotides, at least 2 nucleotides and less than 100 nucleotides, at least 2 nucleotides and less than 50 nucleotides, at least 5 nucleotides and less than 500 nucleotides, at least 5 nucleotides and less than 400 nucleotides, at least 5 nucleotides and less than 300 nucleotides, at least 5 nucleotides and less than 200 nucleotides, at least 5 nucleotides and less than 100 nucleotides, at least 5 nucleotides and less than 50 nucleotides, at least 10 nucleotides and less than 500 nucleotides, at least 10 nucleotides and less than 400 nucleotides, at least 10 nucleotides and less than 300 nucleotides, at least 10 nucleotides and less than 200 nucleotides, at least 10 nucleotides and less than 100 nucleotides, or at least 10 nucleotides and less than 50 nucleotides.

As used herein, the term “variant” “genetic variant” refers to a specific region of the genome which differs between two Mycobacterium tuberculosis genomes. Non-limiting examples include a single-nucleotide polymorphism (SNP), or a mutation, such as an insertion or a deletion. The minor variant detected in the heteroresistant population of M. tuberculosis may be an SNP, an insertion, or a deletion. Non-limiting examples of genetic mutations associated with drug resistance in M. tuberculosis are found in Georghiou et al. (2012) PLoS ONE 7(3):e33275.

As used herein, the term “single-nucleotide polymorphism” of “SNP” refers to a substitution of a single nucleotide that occurs at a specific position in the genome, where each variation is present to some appreciable degree within a population (e.g., >1%).

As used herein, the term “subpopulation” refers to an identifiable fraction or subdivision of a population.

As used herein, the term “read” or “sequence read” refers to the sequence of a cluster that is obtained after the end of the sequencing process which is ultimately the sequence of a section of a unique fragment.

As used herein, the term “micro-heteroresistance” refers to the presence of greater than 0.1% to less than 5% resistant subpopulations in an individual sample, as defined previously¹⁹.

As used herein, the term “pre-resistance” describes samples that have a heteroresistant genotype with susceptible phenotype and subsequently progress to increased levels of genomic heteroresistance or fixed resistance while also attaining phenotypic resistance.

The incidence of drug-resistant (DR) tuberculosis (TB) continues to increase worldwide. Undetected heteroresistance, the presence of DR and susceptible genotypes in bacterial populations involved in infection, at treatment initiation may play a role in the expansion of DR strains and treatment failure. In Mycobacterium tuberculosis (Mtb), current minor DR component detection levels are limited to ˜1%, using phenotypic drug susceptibility testing, which requires 15-30 days or even longer to complete. By that point during an infection, it is likely too late to prevent DR-TB and treatment failure.

Detection of minor components in complex biological mixtures has radically advanced with the emergence of next-generation sequencing. Low-level detection from sequence data, however, is not trivial, primarily due to the error rates in sequencing. The error associated with the respective sequencing platform, as well as the GC content of the organism, sets the limit of discerning actual minor component from error. The use of “single molecule-overlapping reads” (SMOR) analysis, however, for determination of actual mutation ratios in target loci (e.g., antibiotic resistance genes) leads to an increase in heteroresistance detection sensitivity and lower error bias.

The present invention relates to a method of rapidly detecting resistant Mtb subpopulations consisting of 0.1% or less of the total Mtb population, for example in under a week from collection of the sample. With the invention, clinicians can track patient treatment in a timelier fashion and alter the course of treatment when heteroresistance is detected within a week versus a month or more as is common with current technology. This analysis can also be useful to researchers wanting to characterize population structure within a single sample of bacteria. Accordingly, in some aspects, the invention provides a diagnostic assay for the detection of heteroresistance in M. tuberculosis in clinical samples. The present invention may also be used to detect and monitor antibiotic resistance in a subject infected with M. tuberculosis. Antibiotic resistance can be determined by the presence or absence of one or more antibiotic resistance genes or markers in the population. Non-limiting examples of such antibiotic resistance genes include pepQ, Rv0678, ddn, atpE, fbiA, fbiB, fbiC, and fgd.

The method described herein comprises obtaining at least one amplicon from a sample, wherein the at least one amplicon contains a region of interest from Rv0678 (NCBI Reference Sequence: NP_215192.1), pepQ (NCBI Reference Sequence: NP_217051.1), atpE (NCBI Reference Sequence: NP_215821.1), ddn (NCBI Reference Sequence: WP_003419309.1), fbiA (NCBI Reference Sequence: NP_217778.1), fbiB (NCBI Reference Sequence: NP_217779.1), fbiC (NCBI Reference Sequence: NP_215689.1), or fgd (NCBI Reference Sequence: WP_003898438.1); and detecting and/or quantifying a minor variant of the polymorphism. The sample may be collected from sputum, pleural fluid, blood, saliva, or any combination thereof from a subject. The region of interest comprises a polymorphism associated with a drug-resistant subpopulation of Mtb. Thus, detection of the minor variant of the polymorphism indicates the presence of a drug-resistant subpopulation of Mtb in the sample. In some aspects, the drug-resistant subpopulation of Mtb is resistant to bedaquiline-related quinolone derivative and/or nitroimidazole antibiotic (e.g., delamanid). For example, the drug-resistant subpopulation of Mtb in the sample is bedaquiline-resistant and/or nitroimidazole-resistant.

In some aspects, the amplicon consists of less than about 500 nucleotides, less than about 450 nucleotides, less than about 400 nucleotides, less than about 350 nucleotides, less than about 300 nucleotides, less than about 250 nucleotides, less than about 200 nucleotides, less than about 150 nucleotides, less than about 100 nucleotides, or less than about 50 nucleotides.

In some aspects, the region of interest comprises a segment of Rv0678, pepQ, atpE, ddn, fbiA, fbiB, fbiC, or fgd. In some implementations, the amplicon containing the region of interest from Rv0678, pepQ, atpE, ddn, fbiA, fbiB, fbiC, or fgd is produced using at least one primer selected from Table 4. In some embodiments, the amplicon containing the region of interest from Rv0678 is produced using a forward primer and a reverse primer selected from SEQ ID Nos. 20-30, 43, and 44. In other embodiments, the amplicon containing the region of interest from pepQ is produced using a forward primer and a reverse primer selected from SEQ ID Nos. 1-19. In yet other embodiments, the amplicon containing the region of interest from atpE is produced using a forward primer and a reverse primer selected from SEQ ID Nos. 31-34. In other embodiments, the amplicon containing the region of interest from ddn is produced using a forward primer and a reverse primer selected from SEQ ID Nos. 35 and 36. In still other embodiments, the amplicon containing the region of interest from fbiA is produced using a forward primer and a reverse primer selected from SEQ ID Nos. 37, 38 61, 62, 81, and 82. In some embodiments, the amplicon containing the region of interest from fbiB is produced using a forward primer and a reverse primer selected from SEQ ID Nos. 39-42, 57-60, and 77-80. In other embodiments, the amplicon containing the region of interest from fbiC is produced using a forward primer and a reverse primer selected from SEQ ID Nos. 51-56 and 71-76. In still other embodiments, the amplicon containing the region of interest from fgd is produced using a forward primer and a reverse primer selected from SEQ ID Nos. 45-50 and 60-70. In some aspects, the forward and reverse primers used to generate the amplicons containing the region of interest from Rv0678, pepQ, atpE, ddn, fbiA, fbiB, fbiC, or fgd have between X and X nucleotides and comprise a sequence with at least 85% identity to a primer selected from SEQ ID Nos 1-82.

In some embodiments where the region of interest comprises a polymorphism in pepQ associated with the bedaquiline-resistant subpopulation, and the at least one primer used to produce the amplicon comprises a sequence selected from the group consisting of SEQ ID NOs: 1-19. In some embodiments where the region of interest comprises a polymorphism in atpE associated with the bedaquiline-resistant subpopulation, the at least one primer used to produce the amplicon comprises a sequence selected from the group consisting of SEQ ID NOs: 31-34 and 63-64. In some embodiments where the region of interest comprises a polymorphism in ddn associated with the nitroimidazole-resistant subpopulation, the at least one primer used to produce the amplicon comprises a sequence selected from the group consisting of SEQ ID NOs: 35-36. In some embodiments where the region of interest comprises a polymorphism in fbiA associated with the nitroimidazole-resistant subpopulation, the at least one primer used to produce the amplicon comprises a sequence selected from the group consisting of SEQ ID NOs: 37-38, 61-62, and 81-82. In some embodiments where the region of interest comprises a polymorphism in fbiB associated with the nitroimidazole-resistant subpopulation, the at least one primer used to produce the amplicon comprises a sequence selected from the group consisting of SEQ ID NOs: 39-42, 57-60, and 77-80. In some embodiments where the region of interest comprises a polymorphism in fbiC associated with the nitroimidazole-resistant subpopulation, the at least one primer used to produce the amplicon comprises a sequence selected from the group consisting of SEQ ID NOs: 51-56 and 71-76. In some embodiments where the region of interest comprises a polymorphism in fgd associated with the nitroimidazole-resistant subpopulation, the at least one primer used to produce the amplicon comprises a sequence selected from the group consisting of SEQ ID NOs: 45-50 and 65-70.

In some aspects, the minor we of the polymorphism is a SNP, an insertion, a deletion, or any combination thereof. There are characterized SNPs in Mtb that confer resistance to several different antibiotics. Non-limiting examples of genetic mutations associated with drug resistance in M. tuberculosis are found in Georghiou et al. (2012) PLoS ONE 7(3):e33275. In some aspects, the minor variant of the polymorphism is located in the promoter region of a gene or in the genes.

In certain embodiments, the minor variant comprises an insertion or deletion in Rv0678 at position 132, 136, 137, 138, 139, 192, 193, or a combination thereof. For example, the minor variant of the polymorphism in Rv0678 is an insertion of G or GA at position 138, an insertion of T at position 139, an insertion of G at position 192, a deletion of G at position 193, or a combination thereof. In such implementations, the at least one primer used to generate the amplicon comprises a sequence selected from the group consisting of SEQ ID NOs: 20-30 and 43-44. In other embodiments, the minor variant is Rv0678-8 T/G, atpE 201 A/G, or atpE 223 C/T.

In certain aspects, the present invention relates to an approach to applying cutting-edge genomic science and technology to the ongoing clinical and public health problem of multi-drug resistant tuberculosis. In one embodiment, an optimized heteroresistance assay is used to detect known mutations associated with two anti-TB drugs, followed by an evaluation of heteroresistance in serial samples from a patient population. Thus, in certain implementations, the method further comprising predicting phenotypic M. tuberculosis resistance to bedaquiline, nitroimidazole, or both, based on a micro-heteroresistance threshold. The present invention further comprises, in certain embodiment, administering to the subject a regime of antibiotics to effectively control the population of pathogen based on the presence or absence of antibiotic resistance markers in the pathogen.

In some embodiments, a micro-heteroresistance threshold is determined to predict phenotypic M. tuberculosis resistance to bedaquiline, nitroimidazole, or both. In some embodiments the micro-heteroresistance threshold is less than about 5.0%, less than about 4.0%, less than about 3.0%, less than about 2.0%, less than about 1.0%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or greater than about 0.1% of the population.

The minor variant of the polymorphism may be detected using a next generation sequencing (NGS) platform. Accordingly, in some implementations, the method comprises obtaining an amplicon from the sample, wherein the amplicon comprises a region of interest in Rv0678, pepQ, atpE, ddn, fbiA, fbiB, fbiC, fgd, or a combination thereof, and the region of interest comprises a polymorphism associated with the drug-resistant subpopulation; obtaining sequencing data by sequencing the amplicon on a NGS platform; and detecting and/or quantifying a minor variant of the polymorphism, wherein the presence of the minor variant indicates the presence of the drug-resistant subpopulation.

The use of overlapping reads allows for effective coverage of each locus on both strands of an individual sequenced DNA molecule, which in turn allows for an independent confirmation of the specific nucleotide at that single locus. The product rule of probability applies, such that if one locus on a single molecule is read two times, it has the lower limit of detection of the probability of one error occurring squared. In some embodiments, the Illumina Miseq platform is used to sequence amplicons from several different in vitro mixtures of DR and susceptible Mtb strains to validate the use of SMOR for identifying heteroresistance. The calculated average of combined amplification and sequencing error rate for Mtb (a high GC organism) is 0.51% per position across the amplicons tested. When employing SMOR, the theoretical limit of detection of a minor component is 2.6×10⁻⁶, readily allowing for detection of minor components below 0.51%.

By using overlapping reads on targeted regions that contains SNPs conferring resistance to antibiotics, one can characterize heteroresistance in clinical samples down to a level that has not been previously achieved. Overlapping reads have been used in next generation sequencing to improve whole genome examination but they have not been used to add confidence in antibiotic resistance population evaluation.

In some aspects, each of the overlapping nucleic acid strands consists of less than about 500 nucleotides, less than about 450 nucleotides, less than about 400 nucleotides, less than about 350 nucleotides, less than about 300 nucleotides, less than about 250 nucleotides, less than about 200 nucleotides, less than about 150 nucleotides, less than about 100 nucleotides, or less than about 50 nucleotides.

Thus, in some embodiments, the present invention is directed to a next-generation sequencing analysis methodology to detect minor proportions of a sample that contain mutations associated with important phenotypes, including antibacterial resistance. This analysis decreases the sequencing error rate so that extremely low levels of true minor components (e.g., SNP loci) can be detected.

In some embodiments, the limit of detection for the minor variant is less than about 1.0%, less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, less than about 0.1%, less than about 0.09%, less than about 0.08%, less than about 0.07%, less than about 0.06%, less than about 0.05%, less than about 0.04%, less than about 0.03%, less than about 0.02%, or less than about 0.01% of the heteroresistant population.

In certain embodiments, the disclosed method further comprises administering a therapeutic agent to a heteroresistant population of M. tuberculosis. Exemplary therapeutic agents are found in Da Silva et al. (2011) J. Antimicrob. Chemother. 66:1417. Thus in some implementations, the method further comprises treating the subject with an antibiotic or regime of antibiotics. Non-limiting examples of such antibiotics include PA-824, OPC-67683, SQ109, TMC207, NAS-21, NAS-91, and combinations thereof.

Mathematical models of within-host Mtb population dynamics have predicted that heteroresistance can cause the emergence of MDR-TB prior to treatment initiation, and this emergence may occur 1,000-10,000 times more frequently. Studies of within-host dynamics of Mtb growth during treatment of have also indicated that resistant subpopulations can easily dominate a lesion over time in both treatment compliant and non-compliant patients. The presence of resistance conferring mutations, even as minor components of an infecting population of Mtb, likely leads to selection of resistant strains, in the presence of the corresponding drug, and subsequent treatment failure. Minor resistant populations, however, are typically missed through standard analysis of isolates because the dominant organism phenotype masks any minor component variants. In certain aspects, the present invention addresses this problem by providing effective methods to detect and quantify minor resistant populations.

In certain embodiments, the present invention is directed to the detection and analysis of heteroresistance in tuberculosis infections. An assay is provided that is able to accurately detect heteroresistance in Mtb and quantify the presence and proportion of all resistant allele minor components down to less than 0.1% using clinically relevant table-top next generation sequencing (NGS) technology and advanced bioinformatic algorithms. This approach provides a rapid, highly sensitive and specific method for detecting and monitoring the potential clinical relevance of heteroresistance in serial clinical samples from TB patients, which is not achievable by any other existing technology. Additionally, the NGS technology used in the assay can be used for deep sequencing of multiple targeted areas simultaneously, which allows for the detection of extremely rare minor components in a population at all targeted locations at once. This multiplexing approach is ideal for developing a practical, efficient, and rapid analysis of heteroresistance directly from patient sputum, which has significant advantages over existing technologies.

While deep-sequencing seems to be an obvious solution, it is not sufficient, in and of itself. NGS minor variant detection is not trivial; primarily due to the error rates associated with the sequencing platform (e.g. Illumina MiSeq platform has a standard rating of 75% of bases having a 0.1% error). This rate sets a theoretical limit of discerning a rare variant from error but recent advances in technology and bioinformatics allow for minor variant detection at significantly lower levels than expected error rate. An advantage resulting from the approach of the present invention is the ability to accurately detect minor components below the sequencing error by using a “Single-Molecule Overlapping Read” (SMOR) analysis. Thus in some aspects, the detection of minor variant of the polymorphism is performed with a bioinformatics script that requires a user to input genomic regions of interest and generates a report with single molecule-overlapping read information used to identify the minor variant.

The method described herein can detect a 0.3% artificial mixture of SNP alleles in the inhA promoter at a frequency of 3.07×10⁻³, which was at least two orders of magnitude more frequent than identifiable sequence errors. The use of SMOR allows for researchers and clinicians to follow the evolution of heteroresistance, determine its clinical relevance and develop appropriate treatment strategies to suppress minor component resistant sub-populations before they become clinically significant. Thus in some implementations, the method further comprises using a highly homogenous synthetic plasmid standard to identify actual sequence error rate variance between target loci and sequencing runs.

In some aspects, the method of rapidly detecting resistant Mtb subpopulations consisting of 0.1% or less of the total Mtb population comprises providing a sample comprising a population of Mtb; extracting nucleic acids from the sample; amplifying a target locus of the genome Mtb in the extracted nucleic acids, wherein the target locus comprises at least one minor variant associated with drug resistance in Mtb; consecutively sequencing both overlapping nucleic acid strands from a single DNA molecule amplified from the target locus on a NGS platform; applying an alignment algorithm to sequencing data from the overlapping nucleic acid strands; and performing an analysis of the aligned sequencing data to detect the at least one minor variant and heteroresistant population of the Mtn. The at least one minor variant is located in a gene selected from the group consisting of: pepQ, Rv0678, ddn, atpE, fbiA, fbiB, fbiC, and fgd.

In some embodiments, the nucleic acids from the sample are analyzed by Sequencing by Synthesis (SBS) techniques. SBS techniques generally involve the enzymatic extension of a nascent nucleic acid strand through the iterative addition of nucleotides against a template strand. In traditional methods of SBS, a single nucleotide monomer may be provided to a target nucleotide in the presence of a polymerase in each delivery. However, in some of the methods described herein, more than one type of nucleotide monomer can be provided to a target nucleic acid in the presence of a polymerase in a delivery.

SBS can utilize nucleotide monomers that have a terminator moiety or those that lack any terminator moieties. Methods utilizing nucleotide monomers lacking terminators include, for example, pyrosequencing and sequencing using γ-phosphate-labeled nucleotides. In methods using nucleotide monomers lacking terminators, the number of different nucleotides added in each cycle can be dependent upon the template sequence and the mode of nucleotide delivery. For SBS techniques that utilize nucleotide monomers having a terminator moiety, the terminator can be effectively irreversible under the sequencing conditions used as is the case for traditional Sanger sequencing which utilizes dideoxynucleotides, or the terminator can be reversible as is the case for sequencing methods developed by Solexa (now Illumina, Inc.). In preferred methods a terminator moiety can be reversibly terminating.

SBS techniques can utilize nucleotide monomers that have a label moiety or those that lack a label moiety. Accordingly, incorporation events can be detected based on a characteristic of the label, such as fluorescence of the label; a characteristic of the nucleotide monomer such as molecular weight or charge; a byproduct of incorporation of the nucleotide, such as release of pyrophosphate; or the like. In embodiments, where two or more different nucleotides are present in a sequencing reagent, the different nucleotides can be distinguishable from each other, or alternatively, the two or more different labels can be the indistinguishable under the detection techniques being used. For example, the different nucleotides present in a sequencing reagent can have different labels and they can be distinguished using appropriate optics as exemplified by the sequencing methods developed by Solexa (now Illumina, Inc.). However, it is also possible to use the same label for the two or more different nucleotides present in a sequencing reagent or to use detection optics that do not necessarily distinguish the different labels. Thus, in a doublet sequencing reagent having a mixture of A/C both the A and C can be labeled with the same fluorophore. Furthermore, when doublet delivery methods are used all of the different nucleotide monomers can have the same label or different labels can be used, for example, to distinguish one mixture of different nucleotide monomers from a second mixture of nucleotide monomers. For example, using the [First delivery nucleotide monomers]+[Second delivery nucleotide monomers] nomenclature set forth above and taking an example of A/C+(1/T), the A and C monomers can have the same first label and the G and T monomers can have the same second label, wherein the first label is different from the second label. Alternatively, the first label can be the same as the second label and incorporation events of the first delivery can be distinguished from incorporation events of the second delivery based on the temporal separation of cycles in an SBS protocol. Accordingly, a low-resolution sequence representation obtained from such mixtures will be degenerate for two pairs of nucleotides (T/G, which is complementary to A and C, respectively; and C/A which is complementary to G/T, respectively).

Some embodiments include pyrosequencing techniques. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaghi, M., Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) “Real-time DNA sequencing using detection of pyrophosphate release.” Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencing sheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M., Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-time pyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos. 6,210,891; 6,258,568 and 6,274,320, the disclosures of which are incorporated herein by reference in their entireties). In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated is detected via luciferase-produced photons.

In another example type of SBS, cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobleachable dye label as described, for example, in U.S. Pat. Nos. 7,427,67, 7,414,1163 and 7,057,026, the disclosures of which are incorporated herein by reference. This approach is being commercialized by Solexa (now Illumina Inc.), and is also described in WO 91/06678 and WO 07/123,744 (filed in the United States Patent and Trademark Office as U.S. Ser. No. 12/295,337), each of which is incorporated herein by reference in their entireties. The availability of fluorescently-labeled terminators in which both the termination can be reversed and the fluorescent label cleaved facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides.

In other embodiments, Ion Semiconductor Sequencing is utilized to analyze the nucleic acids from the sample. Ion Semiconductor Sequencing is a method of DNA sequencing based on the detection of hydrogen ions that are released during DNA amplification. This is a method of “sequencing by synthesis,” during which a complementary strand is built based on the sequence of a template strand.

For example, a microwell containing a template DNA strand to be sequenced can be flooded with a single species of deoxyribonucleotide (dNTP). If the introduced dNTP is complementary to the leading template nucleotide it is incorporated into the growing complementary strand. This causes the release of a hydrogen ion that triggers a hypersensitive ion sensor, which indicates that a reaction has occurred. If homopolymer repeats are present in the template sequence multiple dNTP molecules will be incorporated in a single cycle. This leads to a corresponding number of released hydrogens and a proportionally higher electronic signal.

This technology differs from other sequencing technologies in that no modified nucleotides or optics are used. Ion semiconductor sequencing may also be referred to as ion torrent sequencing, proton-mediated sequencing, silicon sequencing, or semiconductor sequencing. Ion semiconductor sequencing was developed by Ion Torrent Systems Inc. and may be performed using a bench top machine. Rusk, N. (2011). “Torrents of Sequence,” Nat Meth 8(1): 44-44. Although it is not necessary to understand the mechanism of an invention, it is believed that hydrogen ion release occurs during nucleic acid amplification because of the formation of a covalent bond and the release of pyrophosphate and a charged hydrogen ion. Ion semiconductor sequencing exploits these facts by determining if a hydrogen ion is released upon providing a single species of dNTP to the reaction.

For example, microwells on a semiconductor chip that each contain one single-stranded template DNA molecule to be sequenced and one DNA polymerase can be sequentially flooded with unmodified A, C, G or T dNTP. Pennisi, E. (2010). “Semiconductors inspire new sequencing technologies” Science 327(5970): 1190; and Perkel, J., “Making contact with sequencing's fourth generation” Biotechniques (2011). The hydrogen ion that is released in the reaction changes the pH of the solution, which is detected by a hypersensitive ion sensor. The unattached dNTP molecules are washed out before the next cycle when a different dNTP species is introduced.

Beneath the layer of microwells is an ion sensitive layer, below which is a hypersensitive ISFET ion sensor. All layers are contained within a CMOS semiconductor chip, similar to that used in the electronics industry. Each released hydrogen ion triggers the ISFET ion sensor. The series of electrical pulses transmitted from the chip to a computer is translated into a DNA sequence, with no intermediate signal conversion required. Each chip contains an array of microwells with corresponding ISFET detectors. Because nucleotide incorporation events are measured directly by electronics, the use of labeled nucleotides and optical measurements are avoided.

An example of an Ion Semiconductor Sequencing technique suitable for use in the methods of the provided disclosure is Ion Torrent sequencing (U.S. Patent Application Numbers 2009/0026082, 2009/0127589, 2010/0035252, 2010/0137143, 2010/0188073, 2010/0197507, 2010/0282617, 2010/0300559), 2010/0300895, 2010/0301398, and 2010/0304982), the content of each of which is incorporated by reference herein in its entirety. In Ion Torrent sequencing, DNA is sheared into fragments of approximately 300-800 base pairs, and the fragments are blunt ended. Oligonucleotide adaptors are then ligated to the ends of the fragments. The adaptors serve as primers for amplification and sequencing of the fragments. The fragments can be attached to a surface and are attached at a resolution such that the fragments are individually resolvable. Addition of one or more nucleotides releases a proton (W), which signal detected and recorded in a sequencing instrument. The signal strength is proportional to the number of nucleotides incorporated. User guides describe in detail the Ion Torrent protocol(s) that are suitable for use in methods of the invention, such as Life Technologies' literature entitled “Ion Sequencing Kit for User Guide v. 2.0” for use with their sequencing platform the Personal Genome Machine™ (PCG).

In some embodiments, as a part of the sample preparation process, “barcodes” may be associated with each sample. In this process, short oligos are added to primers, where each different sample uses a different oligo in addition to a primer.

The term “library”, as used herein refers to a library of genome-derived sequences. The library may also have sequences allowing amplification of the “library” by the polymerase chain reaction or other in vitro amplification methods well known to those skilled in the art. The library may also have sequences that are compatible with next-generation high throughput sequencers such as an ion semiconductor sequencing platform.

In certain embodiments, the primers and barcodes are ligated to each sample as part of the library generation process. Thus, during the amplification process associated with generating the ion amplicon library, the primer and the short oligo are also amplified. As the association of the barcode is done as part of the library preparation process, it is possible to use more than one library, and thus more than one sample. Synthetic DNA barcodes may be included as part of the primer, where a different synthetic DNA barcode may be used for each library. In some embodiments, different libraries may be mixed as they are introduced to a flow cell, and the identity of each sample may be determined as part of the sequencing process. Sample separation methods can be used in conjunction with sample identifiers. For example, a chip could have 4 separate channels and use 4 different barcodes to allow the simultaneous running of 16 different samples.

Also described are primer and kits for detecting and/or quantifying a drug-resistant subpopulation of M. tuberculosis in a sample. The primer comprises a sequence with at least 85% identity to a sequence set forth in SEQ ID NOs: 1-82 and a label, wherein the primer is between 10 to 70 nucleotides in length. In some embodiments, the primer consists of a sequence set forth in SEQ ID NOs: 1-82. The kit comprises at least one of the primers described herein. In some embodiments, the kit further comprises reagents for amplification of a genomic sample.

Examples

The present invention is further illustrated by the following examples that should not be construed as limiting. The contents of all references, patents, and published patent applications cited throughout this application, as well as the Figures, are incorporated herein by reference thereto in their entirety for all purposes.

Targeted Deep Sequencing Identifies the Amplification of Bedaquiline Micro-Heteroresistance Following Tuberculosis Treatment Cessation.

1. Methods

a. Whole Genome Sequencing (WGS) of Serial Patient Isolates

Stored clinical isolates spanning most of the course of treatment and several months after cessation of treatment were re-cultured, and DNA was extracted as previously described¹. Eight isolates were available for WGS using the Illumina NextSeq platform at a median coverage of 62x. Sequencing reads were mapped to a reference genome (Mycobacterium tuberculosis, H37Rv, Genbank: AL123456) with variant calling and annotation performed as previously described. All identified variants were visually inspected using Tablet³. Also, TB profiler⁴ was used to ascertain the genotypic drug susceptibility (DST) profile. Raw sequencing reads have been deposited at the European Nucleotide Archive. All variants identified in Rv0678 were confirmed by Sanger sequencing using the following primer pair (forward primer: 5′ AGAGTTCCAATCATCGCCCT 3′ (SEQ ID NO: 169); reverse primer: 5′ TGCTCATCAGTCGTCCTCTC 3′ (SEQ ID NO: 170)).

b. Targeted Deep Sequencing and Data Analysis

Targeted deep sequencing was done as previously described⁵ using the following primer pair; forward primer: 5′ ACCCAACTGAATGGAGCGAAACTTGTGAGCGTCAACGACG 3′ (SEQ ID NO: 171); reverse primer: 5′ ACGCACTTGACTTGTCTTCGGTTGCTCATCAGTCGTCCT 3′ (SEQ ID NO: 172). Data analysis was done using the Allele-specific alignment pipeline (ASAP)⁵ using two aligners, Bowtie⁶ and BWA⁷. Only short insertions and deletions identified in both alignment files generated by BWA and Bowtie were included for further analysis.

c. Bedaquiline Phenotypic Drug Susceptibility Testing

Phenotypic DST for bedaquiline was conducted using the BACTEC MGIT 960 system and EpiCentre software equipped with the TB eXist module for DST⁸. Briefly, each M. tuberculosis isolates was subcultured in MGIT supplemented with Oleic Albumin Dextrose Catalase (OADC) until a positive growth was observed. Thereafter 0.5 mL of each subculture was added to respective MGIT tubes supplemented with 0.8 mL of OADC and containing a final bedaquiline concentration of 1 μg/ml. Concurrently, a growth control was prepared by inoculating 0.5 mL of a 1:100 dilution of each subculture into respective MGIT tubes supplemented with 0.8 mL of OADC without bedaquiline. The laboratory strain, H37Rv (ATCC 27294), was used as a susceptible control and the M. tuberculosis strain (BCCM/ITM 121749), obtained from Belgium coordinated collection of micro-organisms (A63P mutation in atpE) was used as a resistant control. Isolates were considered resistant if the growth index of the bedaquiline containing tube was greater than 100 when the growth control reached a growth index of 400.

Bedaquiline improves survival among individuals with multidrug-resistant tuberculosis (MDR-TB)^(9,10). The inventors report a 65-year old HIV-negative South African male diagnosed in 2013 with MDR-TB (fluoroquinolone and amikacin phenotypically susceptible) using Xpert MTB/RIF and Genotype MTBDRplus. Baseline X-ray showed bilateral TB disease with left apex cavitation, right hilar infiltrate, and consolidation in the right apex. Genotype MTBDRplus showed low-level isoniazid resistance.

2. Chronology of the Treatment

FIG. 1A depicts the chronology of the treatment of the case study and includes a summary of treatment provision. Standardized treatment including moxifloxacin (MXF), pyrazinamide (Z), kanamycin (KAN), ethionamide (ETH), isoniazid (INH), and terizidone (TZD), as per national guidelines within two days of diagnosis, was initiated. After initial sputum culture conversion (month 3) and clinical improvement, the patient reconverted to culture positive and developed bilateral cavitation. Isoniazid was withdrawn 28 days after treatment initiation. Kanamycin was stopped 6 months after treatment initiation.

Following detection of phenotypic ofloxacin resistance (month 6), treatment was revised (month 8) to include high-dose isoniazid (hdIND, 800 mg), ethambutol (E), pyrazinamide (Z), terizidone (TZD), linezolid (LZD), para-aminosalicylic acid (PAS), and kanamycin (KAN). Bedaquiline (BDQ) was added 22 days later and administered for a total of 27 weeks per the South African Bedaquiline Clinical Access Program¹¹. The patient was admitted to a TB inpatient facility for the first two months of BDQ treatment. By examination of the patient's treatment card and patient interview adherence to bedaquiline during both in-patient and out-patient treatment was subjectively assessed as good, although strict direct observation of treatment was not practiced. Pyrazinamide (Z) and ethambutol (E) were stopped at 2.3 months following revised regimen initiation due to persistent arthralgias and changes in vision. The patient refused kanamycin (KAN) at month 6 for a duration of 2.4 months after more than 12 months of injectable treatment. Kanamycin (KAN) and high-dose isoniazid (hdIND, due to vision problems) were stopped at 13 months.

The patient remained culture positive (treatment failure). The physician decided to stop all treatment at 15.7 months after initiation of the revised regimen, after which the patient was transitioned to palliative care and died 7 months later.

2. Phenotype Characterization

As per guidelines, isolates were phenotypically characterized for ofloxacin and amikacin susceptibility. Isolates taken at diagnosis and initiation of treatment were culture-positive and susceptible to ofloxacin and amikacin based on routine phenotypic DST (National Health Laboratory Services, Green Point, South Africa). Follow-up routine sputum specimens were taken 42 days and 3.4 months after treatment initiation were acid-fast bacilli (AFB) smear and culture negative (Table 1). Four subsequent sputum specimens collected 4 to 8 months after treatment initiation were smear- and culture-positive. The sputum specimen taken 6 months after initiation of standard treatment showed phenotypic resistance to ofloxacin using phenotypic DST, and the patient was classified as having had treatment failure. Month 6 chest x-ray showed extensive fibrosis in the left lung and cavitation in both apices. All isolates remained susceptible to second-line injectables. The individualized regimen was continued until the outcome of treatment failure at 15 months. All sputum cultures after the stopping of all treatment were positive. All isolates with a variant frequency of >1% in Rv0678 were resistant to bedaquiline at 1 μg/ml in MGIT.

3. Genotypic Drug Resistance

FIGS. 2-3 depict genotypic drug resistance (based on WGS), phenotypic bedaquiline drug susceptibility testing (DST, MGIT), targeted deep sequencing and treatment monitoring during standardized treatment, and a subsequent individualized bedaquiline-containing regimen. During the course of treatment, a total of 19 sputum cultures were requested (Table 1), of which eight could be retrieved from the National Health Laboratory Service (NHLS) in Cape Town for next-generation sequencing (NGS). Overall, eight M. tuberculosis isolates (A-H) collected 4.7 months after initiation of standard treatment regimen until 6 months after all TB treatment was stopped underwent whole-genome sequencing (WGS), targeted deep sequencing (TDS)⁵ of Rv0678, and phenotypic bedaquiline drug susceptibility testing (DST). All eight isolates differed by a maximum of five variants, implying in vivo evolution rather than reinfection with a different strain.

Isolate A WGS of the first available isolate (isolate A) collected 4.7 months after initiation of standard MDR-TB treatment regimen revealed that the patient was infected with a Beijing strain. The strain harbored mutations in rpoB (S450L), inhA promoter region (−15 T/C), embB (M306V), ethA (65 T insertion), ethR (A95T), gyrA (D94G), pncA (467 GCACCC deletion), and rrs (S514R), associated with resistance to rifampicin, isoniazid, ethambutol, ethionamide, fluoroquinolones, pyrazinamide and streptomycin, respectively. All of these resistance-causing mutations were present in 100% of the sequencing reads. The detection of a D94G substitution in gyrA, which confers resistance to fluoroquinolones, suggests that the classification of fluoroquinolone susceptibility by phenotypic DST performed at the routine laboratory on the same isolate was incorrect.

Isolate B The isolate was taken 7.2 months after treatment initiation (isolate B) did not show amplification of resistance but showed the loss of a variant in rpoC (A734G) (Table 2, FIG. 3). According to the WGS data, the patient received only two potentially effective drugs (kanamycin and terizidone). Phenotypic resistance to ofloxacin was first detected by the routine laboratory on the specimen collected 6 months after treatment initiation. Targeted deep sequencing (TDS)⁴ of isolate B did not detect any underlying variants in Rv0678⁵, the gene associated with resistance to bedaquiline¹².

Isolate C WGS of isolate C was collected 2 months after treatment revision, i.e., 2 months after initiation of the individualized pre-XDR treatment regimen and 10 weeks after the initiation of bedaquiline. WGS showed the presence of wild-type sequences for the genes associated with resistance to second-line injectables (rrs (1401 region)), PAS (thyA, folC, dfrA, ribD), linezolid (rrl, rplC), terizidone (ddl, cycA, alr, ald) and bedaquiline (Rv0678) suggesting that the patient likely received five effective anti-TB drugs (high dose isoniazid, kanamycin, linezolid, terizidone, and PAS) at the time of addition of bedaquiline.

The isolate was phenotypically susceptible to bedaquiline. Despite phenotypic susceptibility to bedaquiline and genotypic susceptibility on WGS, TDS, however, showed the presence of micro-heteroresistance against bedaquiline. There is a C insertion (ins) at position 192 in Rv0678⁵ in 0.05% of the reads (Table 3). This was not present in isolate B taken before the inclusion of bedaquiline.

Isolate D In the isolate collected one week after bedaquiline treatment was stopped (isolate D), WGS and TDS showed that the Rv0678 192 insertion was fixed, present in >90% of the bacterial population. TDS showed the presence of multiple low frequency (>0.1%) indels (insertions and deletions) in codons 194 to 198 in Rv0678. WGS also showed a fixed variant in Rv2839c (S347P) (Table 2). Isolate D was also phenotypically resistant to bedaquiline.

Isolates E-H Subsequently, the 192 C insertion decreased to 0.1% of reads in isolate F (taken 12.3 months after the start of the individualized regimen and 5 months after the cessation of bedaquiline) and was replaced with a different Rv0678 variant (GA insertion at gene position 138) and a second rpoB variant (S582A) according to WGS. In the subsequent isolates taken 15.7 months (isolate G) and 21.7 months (isolate H) after initiation of the revised regimen, WGS and TDS showed the systematic decrease of the Rv0678 138 GA insertion over time and the gain of a third Rv0678 variant (G insertion at position 138). WGS showed the emergence of a rpoC (V483G) variant in this isolate. The G insertion at position 138 became fixed after all treatment was stopped (isolates G and H). The Rv0678 138 G insertion and rpoC V483G were found with a variant frequency of 96% in the last isolate taken. The systematic gain and loss of Rv0678 variants identified by WGS were confirmed by Sanger sequencing (data not shown). All isolates with a variant frequency of >1% in Rv0678 were resistant to bedaquiline at a concentration of 1 μg/ml in MGIT media. Isolates D, E, F, G, and H were phenotypically resistant to bedaquiline.

This case demonstrates the rapid acquisition of bedaquiline resistance despite the presence of five potentially effective drugs and presumed good adherence. These data also highlight the potential utility of sequencing approaches to guide treatment and monitor resistance emergence and the need to administer effective regimens from the start.

The emergence of Rv0678 variants, after completion of 6 months of bedaquiline treatment, demonstrates the risk of resistance amplification after cessation of a drug with a long half-life (5.5 months for bedaquiline)¹³. The identification of a subpopulation of bacilli harboring a variant in Rv0678 ten weeks after the addition of bedaquiline suggests that bedaquiline resistance emerged soon after its inclusion in the revised MDR-TB regimen and was subsequently selected as treatment continued. Following the withdrawal of bedaquiline, a further gain and loss of Rv0678 variants were observed over the course of the long half-life of bedaquiline (5.5 months) Without wishing to be bound by theory; these results suggest that new Rv0678 variants can emerge while plasma concentration of bedaquiline is decreasing.

Alternatively, the variants could have emerged in different lesions prior to the withdrawal of bedaquiline and subsequently observed as these lesions ruptured into the airways. The data also suggests that bedaquiline resistance in this patient developed despite treatment with a background regimen containing five anti-TB drugs that were likely effective based on susceptibility. This highlights a lack of comprehensive understanding of resistance emergence during treatment. No amplification of resistance causing mutations was identified in the serial patient isolates, suggesting that the isolates would still be susceptible to those drugs (high dose isoniazid, kanamycin, linezolid, terizidone, and PAS). The presence of bedaquiline-resistant M. tuberculosis following cessation of bedaquiline and cessation of all TB treatment because of treatment failure poses a transmission risk and threatens the longevity of this new drug. Monitoring of pre-existing and emerging bedaquiline resistance should be a priority among patients with delayed sputum culture conversion and those with positive sputum cultures post bedaquiline cessation.

Four of the nine variable loci were in rpoB and rpoC genes, associated with rifampicin resistance or fitness compensatory mechanisms. Secondary rpoB mutations have been shown to improve growth characteristics and fitness rates in a BCG model¹⁴. Numerous studies have investigated the role of rpoC mutations in compensating for the loss of fitness due to rpoB mutations¹⁵⁻¹⁸. Three of the four variants were however transient, and only one variant (V483G) was fixed in the last available isolate. We have reported the V483G substitution to be the most frequent rpoC variant in our setting, which is also associated with transmission clusters as defined by IS6110 fingerprinting¹⁶.

In summary, this case demonstrates the rapid acquisition of bedaquiline resistance in the presence of five likely effective drugs. There was no evidence of poor adherence to treatment over this time. The emergence of Rv0678 variants, after completion of 6 months bedaquiline, demonstrates the risk of resistance amplification after cessation of a drug with a long half-life (5.5 months for bedaquiline)¹¹. These data highlight the potential utility of sequencing approaches to guide treatment and monitor resistance emergence and the need to incorporate new drugs into more effective regimens from the start of treatment.

4. High Frequency of Bedaquiline Resistance in Programmatically Treated Drug-Resistant TB Patients with Sustained Culture-Positivity in Cape Town, South Africa

Potentially transformative new TB drugs like bedaquiline are undergoing roll-out; however, this is largely in the absence of programmatic DST. Information is lacking on how susceptibility changes during treatment in patients on bedaquiline-containing regimens, especially in at risk patients who have complex treatment histories, are in programmatic rather than trial environments, and have a delayed treatment response (defined here as sustained culture-positivity).

Serial isolates from 51 patients with drug resistant (DR-) TB who were culture-positive after ≥4 months of a programmatically administered bedaquiline-containing regimen, were collected. Bedaquiline phenotypic DST in MGIT 960 (1 μg/ml), targeted deep sequencing (Rv0678, atpE, pepQ) and whole genome sequencing was done on paired isolates (pre-bedaquiline initiation, post-four-month). 24/51 (47%) patients were phenotypically and genotypically resistant (39% acquired resistance). Excluding one patient with an unknown history, prior clofazimine exposure was associated with bedaquiline-resistance [21/24 (88%) bedaquiline resistant cases had prior clofazimine vs. 12/26 (46%) susceptible; p=0.002]. Diverse combinations of single SNPs and indels were in the Rv0678 promoter region and the Rv0678 and atpE genes. Examples of newly described resistance associated variants (RAVs) include Rv0678-8 T/G and atpE 223 C/T. RAVs were not in defined hotspots and sometimes occurred concurrently with atpE RAVs.

The rate of bedaquiline resistance acquisition in this population is alarmingly high and associated with prior clofazimine exposure. The diverse RAVs pose challenges to molecular test development. This study highlights the existence of a potentially infectious pool of bedaquiline-resistant patients present under programmatic conditions in a resource-constrained setting and illustrates the danger of starting patients with complex histories on a novel drug without routinely available DST.

TABLE 1 M. tuberculosis Isolates Collected Over the Course of Treatment Smear GenoType MTBDRplus Phenotypic DST WGS Isolate taken microscopy Culture Rifampicin Isoniazid Amikacin Ofloxacin isolate Time after initiation of   0 days* Neg Pos R R S S treatment   2 days 2+ Pos R R S S   41 days Neg Neg  3.4 months Neg Neg  4.7 months Scanty Pos R R S S A  6.2 months 3+ Pos R R S R  7.2 months Scanty Pos R R S S B   8 months 3+ Pos R R S R Time after initiation of   20 days 3+ Pos R R S S revised MDR regimen   22 days 3+ Pos R R S S   2 months Scanty Pos R R S S C  3.5 months Neg Neg  5.6 months Neg Pos ND ND  7.2 months Neg Pos R R S S D  9.7 months Scanty Pos R R S R E 10.3 months Neg NTM 12.3 months Neg Pos R R S R F 15.7 months Neg Pos R R S R G 21.7 months 3+ Pos R R S R H MTB = M. tuberculosis; WGS = whole genome sequencing; Pos = positive; Neg = negative; R = resistant; S = sensitive; ND = not done; NTM = non-tuberculosis mycobacteria *Xpert MTB/RIF MTB complex positive and rifampicin resistant

TABLE 2 Variants Identified Through Whole-Genome Sequencing in Serial Isolates Cultured from the Patient During Treatment. Time relative Variant frequency of variants identified by whole genome sequencing* to the initiation Rv0678 Rv0678 of the revised rpoC 192 G Rv2839c rpoB 138 GA 138 G rpoC Rv3777 Isolate regimen A734G L823M ins S347P S582A ins ins V483G 147 Syn A −102 86 0 0 0 0 0 0 0 0 days (14/85) B −29 days 0 0 0 0 0 0 0 0 0 C 62 0 100 0 0 0 0 0 0 0 days (36/36) D 7.2 0 0 100 100 0 0 0 0 0 months (56/56) (56/56) E 9.7 0 0 100 100 0 0 0 0 0 months (56/56) (56/56) F 12.3 0 0 0 0 100 100 0 0 0 months (81/81) (75/75) G 15.7 0 0 0 0 35 25 63 65 52 months (24/67) (18/71) (45/71) (52/79) (36/68) H 21.7 0 0 0 0 0 0 96 95 91 months (79/82) (65/68) (73/80) *In brackets-Number of reads with the minor variant/total number of reads

TABLE 3 Minority Populations Identified Through Targeted Deep Sequencing of Rv0678 138 G insertion 138 GA insertion 139 T insertion 192 G insertion 193 G deletion Isolate Date^(#) WGS* TDS* WGS* TDS* WGS* TDS* WGS* TDS* WGS* TDS* A −102 days 0 — 0 — 0 — 0 — 0 — B  −29 days 0 0 0 0 0 0 0 0 0 0 C 62 0 0 0 0 0 0 0 0.05 0 0 days (7/15886) D^($) 7.2 0 0 0 0 0 0 100 96.66 0 0 months (56/56) (17551/18158) E 9.7 0 — 0 — 0 — 100 — 0 — months (56/56) F 12.3 0 0 100 97.52 0 0 0 0.1 0.37 0 months (75/75) (13299/13638) (13/13638) (50/13638) G 15.7 63 65.48 25 28.35 0 0 0 3.22 0.28 0 months (45/71) (9317/14230) (18/71) (4034/14230) (461/14230) (39/14230) H 21.7 96 91.68 0 5.86 0 0.14 0 0.03 0 0 months (79/82) (13029/14212) (832/14212) (19/14212) (4/14212) ins = insertion; WGS = whole genome sequencing; TDS = targeted deep sequencing; “—” = not done ^(#)relative to initiation of revised MDR-TB regimen; *In brackets-Number of reads with the minor variant/total number of reads ^($)other low frequency variants identified in isolate D: 194 T insertion (0.32%), 195 C insertion (0.22%), 196 T insertion (0.22%), 197 T insertion (0.18%), 198 T insertion (0.17%)

TABLE 4 Non-limiting Examples of the Oligonucleotide Sequences Useful for Detecting Single Nucleotide Polymorphisms (SNP) Associated with Drug Resistance RDST or SEQ Drug Gene SMOR Primer name Oligonucleotide sequence ID NO: Bedaquiline pepQ RDST pepQ_UT_F1 ACCCAACTGAATGGAGCGTTGATCA 1 ATGCCCCCTGGAACAG Bedaquiline pepQ RDST pepQ_UT_R1 ACGCACTTGACTTGTCTTCTTTAACC 2 TCGCGCAGTGACTCCACA Bedaquiline pepQ RDST pepQ_UT_F2 ACCCAACTGAATGGAGCGTTCGATAT 3 GACCCGCACCTTCGT Bedaquiline pepQ RDST pepQ_UT_R2 ACGCACTTGACTTGTCTTCCCGAGAG 4 CACGTTCTTCAACTTGG Bedaquiline pepQ SMOR pepQf-60 ACCCAACTGAATGGAGCCAACCCGC 5 GCAGCATCCAGTTAGTCAT Bedaquiline pepQ SMOR pepQf74 ACCCAACTGAATGGAGCCGACCTGA 6 TAAACGTGCGATATCTATCAGGCTTC Bedaquiline pepQ SMOR pepQf199 ACCCAACTGAATGGAGCAAGCGCCC 7 GACCTCGAAGTGGC Bedaquiline pepQ SMOR pepQf351 ACCCAACTGAATGGAGCAACACCGA 8 GTTGGTGCGGGCATCC Bedaquiline pepQ SMOR pepQf496 ACCCAACTGAATGGAGCGCCGAACC 9 GAACGGCAGGTGAGC Bedaquiline pepQ SMOR pepQf640 ACCCAACTGAATGGAGCCGGCGATT 10 TCGTGAAGATCGACTTCGG Bedaquiline pepQ SMOR pepQf899 ACCCAACTGAATGGAGCGCAGATAC 11 ATGAAGCGCCGGGCATC Bedaquiline pepQ SMOR pepQr152 ACGCACTTGACTTGTCTTCCGCGCTC 12 ATCGGCGAACACCA Bedaquiline pepQ SMOR pepQr324 ACGCACTTGACTTGTCTTCGTCCAGG 13 CCGTCCACCGTGACCA Bedaquiline pepQ SMOR pepQr421 ACGCACTTGACTTGTCTTCCCAGCTC 14 GCCGGCGTCTTTAACCT Bedaquiline pepQ SMOR pepQr535 ACGCACTTGACTTGTCTTCGGGCCTC 15 CAGCTCGCGGCTCAC Bedaquiline pepQ SMOR pepQr713-1 ACGCACTTGACTTGTCTTCACGAAGG 16 TGCGGGTCATATCGGAGTGGTA Bedaquiline pepQ SMOR pepQr713-2 ACGCACTTGACTTGTCTTCACGAAGG 17 TGTGGGTCATATCGcAGTGGTA Bedaquiline pepQ SMOR pepQr968 ACGCACTTGACTTGTCTTCGTCACCA 18 CGGAGCCCGCCAGTAGTGTA Bedaquiline pepQ SMOR pepQr+19 ACGCACTTGACTTGTCTTCTGGTCGC 19 CACGTGGGTCTCCTACAGA Bedaquiline Rv0678 SMOR Rv0678f-57 ACCCAACTGAATGGAGCCACGCCGG 20 TCTGGTGACGCATACC Bedaquiline Rv0678 SMOR Rv0678f64 ACCCAACTGAATGGAGCAGATGGGC 21 GGCTATTTCGAGTCCAGGAGTT Bedaquiline Rv0678 SMOR Rv0678f115-1 ACCCAACTGAATGGAGCTGTTGGGCT 22 GGCTGCTGGTGTGTGAT Bedaquiline Rv0678 SMOR Rv0678f115-2 ACCCAACTGAATGGAGCTATTGGGCT 23 GGtTGCTGGTGTGTGAT Bedaquiline Rv0678 SMOR Rv0678f291-1 ACCCAACTGAATGGAGCAACGCTTTC 24 GCGGCTGGCGAG Bedaquiline Rv0678 SMOR Rv0678f291-2 ACCCAACTGAATGGAGCAATGCTTTC 25 GCGGCCGGCGAG Bedaquiline Rv0678 SMOR Rv0678r124 ACGCACTTGACTTGTCTTCAGCCCAA 26 CAATCGACCCGCCAACC Bedaquiline Rv0678 SMOR Rv0678r255 ACGCACTTGACTTGTCTTCGACCGCG 27 AGCCGCTCAATGAACC Bedaquiline Rv0678 SMOR Rv0678r336-1 ACGCACTTGACTTGTCTTCGGCCATT 28 GCCCGGATGCGTTCA Bedaquiline Rv0678 SMOR Rv0678r336-2 ACGCACTTGACTTGTCTTCGGCCATc 29 GCCCGGATGCGcTCA Bedaquiline Rv0678 SMOR Rv0678r+24 ACGCACTTGACTTGTCTTCTCGGTCA 30 GATTGCGAGGTTGCTCATCA Bedaquiline atpE SMOR atpEf-84 ACCCAACTGAATGGAGCAGCCAAGC 31 GATGGAGCTCGAAGAGGAAC Bedaquiline atpE SMOR atpEf130 ACCCAACTGAATGGAGCAGGCGCAA 32 GGGCGGCTGTTCACA Bedaquiline atpE SMOR atpEr222 ACGCACTTGACTTGTCTTCGAACAGC 33 GCCATAAAMGCCAGGTTGATG Bedaquiline atpE SMOR atpEr+96 ACGCACTTGACTTGTCTTCGCTGGAC 34 TCGCCGCCTTCCTCTGC Nitroimidazole ddn RDST ddnF ACCCAACTGAATGGAGCTTGGTCGCT 35 AGGATCAGCGTC Nitroimidazole ddn RDST ddnR ACGCACTTGACTTGTCTTCTCGGCGA 36 AGTTGGGAACGG Nitroimidazole fbiA RDST fbiAF1 ACCCAACTGAATGGAGCGCGCCGGG 37 AGGTACTGTC Nitroimidazole fbiA RDST fbiAR1 ACGCACTTGACTTGTCTTCGCTTTCG 38 TCGACCGGGTCGG Nitroimidazole fbiB RDST fbiBF3 ACCCAACTGAATGGAGCAACTGTTG 39 CGCAGGTCCGTT Nitroimidazole fbiB RDST fbiBR3 ACGCACTTGACTTGTCTTCCAGCAAG 40 GCTTGTACGGCC Nitroimidazole fbiB RDST fbiBF1 ACCCAACTGAATGGAGCCGCTGCTG 41 ATGACCGACCC Nitroimidazole fbiB RDST fbiBR1 ACGCACTTGACTTGTCTTCCGTCCCA 42 TGGTGTCGGTGA Nitroimidazole fgd RDST Fgd2_F2 ACCCAACTGAATGGAGCGGCTTGGG 43 CGATCCAACCATC Nitroimidazole fgd RDST Fgd2_R2 ACGCACTTGACTTGTCTTCGGCCATT 44 CGATGTTTCCCTGGC Nitroimidazole fgd RDST Fgd1_F3 ACCCAACTGAATGGAGCGCTGTCGTT 45 GACAGCTGAGCA Nitroimidazole fgd RDST Fgd1_R3 ACGCACTTGACTTGTCTTCCAAAAGC 46 CCCAGTGCAATCGTC Nitroimidazole fgd RDST Fgd1_F1 ACCCAACTGAATGGAGCCGCGTTTAT 47 GGCATAGGAGTAG Nitroimidazole fgd RDST Fgd1_R1 ACGCACTTGACTTGTCTTCCGTCAAA 48 GTCGACGCGGTCA Nitroimidazole fbiC RDST FbiC_F6 ACCCAACTGAATGGAGCGTGGGTCT 49 GCGGTCATCATC Nitroimidazole fbiC RDST FbiC_R6_redo ACGCACTTGACTTGTCTTCTAGGCCG 50 CAAGCAGGGCGT Nitroimidazole fbiC RDST FbiC_F4 ACCCAACTGAATGGAGCATCTGGGC 51 GCAGCGATCGA Nitroimidazole fbiC RDST FbiC_R4 ACGCACTTGACTTGTCTTCCCGGTGA 52 CCGGTAGCTCGG Nitroimidazole fbiC RDST FbiC_R2 ACGCACTTGACTTGTCTTCCATCGCG 53 GTGTGTTCCTTGG Nitroimidazole fbiC RDST FbiC_F2 ACCCAACTGAATGGAGCTTCGGTTGC 54 AAGGAAGCGC Nitroimidazole fbiB RDST FbiB_F3 ACCCAACTGAATGGAGCCAACTGTT 55 GCGCAGGTCCGTT Nitroimidazole fbiB RDST FbiB_R3 ACGCACTTGACTTGTCTTCCAGCAAG 56 GCTTGTACGGCC Nitroimidazole fbiB RDST FbiB_F1 ACCCAACTGAATGGAGCCGCTGCTG 57 ATGACCGACCC Nitroimidazole fbiB RDST FbiB_R1 ACGCACTTGACTTGTCTTCCGTCCCA 58 TGGTGTCGGTGA Nitroimidazole fbiA RDST FbiA_F1 ACCCAACTGAATGGAGCGCGCCGGG 59 AGGTACTGTC Nitroimidazole fbiA RDST FbiA_R1 ACGCACTTGACTTGTCTTCGCTTTCG 60 TCGACCGGGTCGG Nitroimidazole fgd RDST Fgd2_F3 ACCCAACTGAATGGAGCCCCACCAA 61 CGCCAGGGTC Nitroimidazole fgd RDST Fgd2_R3 ACGCACTTGACTTGTCTTCCAGGATG 62 CACTCTCGAAGGTGTGC Nitroimidazole fgd RDST Fgd2_F1 ACCCAACTGAATGGAGCGTAGGTGC 63 GGTCTAGCGGCT Nitroimidazole fgd RDST Fgd2_R1 ACGCACTTGACTTGTCTTCCACCCTG 64 GCCGGCCGATAC Nitroimidazole fgd RDST Fgd1_F2 ACCCAACTGAATGGAGCCCGAACCG 65 TGTTTTCCTTGGC Nitroimidazole fgd RDST Fgd1_R2 ACGCACTTGACTTGTCTTCCGGGTCG 66 TCGATGCTGTGCTTC Nitroimidazole fbiC RDST FbiC_F5 ACCCAACTGAATGGAGCCCGGGGCC 67 ACCGAAGTAT Nitroimidazole fbiC RDST FbiC_R5 ACGCACTTGACTTGTCTTCTTAAGAT 68 GGGCGACCCAGTGCCG Nitroimidazole fbiC RDST FbiC_F3 ACCCAACTGAATGGAGCTGATCGTG 69 CAGAACTTCCGCG Nitroimidazole fbiC RDST FbiC_R3 ACGCACTTGACTTGTCTTCGCGGTAT 70 TGCGGCCCTGAGT Nitroimidazole fbiC RDST FbiC_F1 ACCCAACTGAATGGAGCGCAGGGAA 71 GGTATACCAACGTG Nitroimidazole fbiC RDST FbiC_R1 ACGCACTTGACTTGTCTTCCCGTTCG 72 CCGAGCCATTCG Nitroimidazole fbiB RDST FbiB_F4 ACCCAACTGAATGGAGCCACACAGC 73 TACCCCGATGCC Nitroimidazole fbiB RDST FbiB_R4 ACGCACTTGACTTGTCTTCGGTAGCC 74 TATCGTCGCTAGAGCG Nitroimidazole fbiB RDST FbiB_F2 ACCCAACTGAATGGAGCAGCGGCTC 75 GGCGTCAC Nitroimidazole fbiB RDST FbiB_R2 ACGCACTTGACTTGTCTTCCCGCAGC 76 CTCGACGAGGTC Nitroimidazole fbiA RDST FbiA_F2 ACCCAACTGAATGGAGCTTGCGAAA 77 CCCATGTAGTGATCA Nitroimidazole fbiA RDST FbiA_R2 ACGCACTTGACTTGTCTTCACCATCT 78 CAGCCGTCGCGTTC Nitroimidazole fbiA SMOR Mtb_fbiAF-70-UT1 ACCCAACTGAATGGAGCGTCCCGGC 79 GTGTCGAGCGTGACTC Nitroimidazole fbiA SMOR Mtb_fbiAR213-UT2 ACGCACTTGACTTGTCTTCGCCGCCC 80 AGGGTATACATGCAGGTGTC Nitroimidazole fbiA SMOR Mtb_fbiAF313-UT1 ACCCAACTGAATGGAGCGGGACCGC 81 GATCTGGCYACCCAT Nitroimidazole fbiA SMOR Mtb_fbiAR600-UT2 ACGCACTTGACTTGTCTTCGATCGCT 82 TCGGTTGCAGCGCTGGAC Nitroimidazole fbiA SMOR Mtb_fbiAF437-UT1 ACCCAACTGAATGGAGCCGACGACC 83 GTTGCGAAACCCATGTAGTG Nitroimidazole fbiA SMOR Mtb_fbiAR665-UT2 ACGCACTTGACTTGTCTTCGCGCCGA 84 TGCTGACCACCGGATTAG Nitroimidazole fbiA SMOR Mtb_fbiAF630-UT1 ACCCAACTGAATGGAGCCTGGCGCC 85 GTCTAATCCGGTGGTCAG Nitroimidazole fbiA SMOR Mtb_fbiAR895-UT2 ACGCACTTGACTTGTCTTCCAATCTC 86 AGCGTGGTCGCCGTCGTG Nitroimidazole fbiA SMOR Mtb_fbiAF80-UT1 ACCCAACTGAATGGAGCGTTTGCTGC 87 CAATTCTGCCCACTCGGAC Nitroimidazole fbiA SMOR Mtb_fbiAR372-UT2 ACGCACTTGACTTGTCTTCGGGGTAG 88 CCGGCCTGCAGCATCTG Nitroimidazole fbiA SMOR Mtb_fbiAF839-UT1 ACCCAACTGAATGGAGCCGCCACCG 89 GGATACTGGACTGCTGG Nitroimidazole fbiA SMOR Mtb_fbiAR+13-UT2 ACGCACTTGACTTGTCTTCTGTTCGG 90 GGCCGGTCAAGCTACCACTC Nitroimidazole fbiB SMOR Mtb_fbiBF-10-UT1 ACCCAACTGAATGGAGCGAGTGGTA 91 GCTTGACCGGCCCCGAACA Nitroimidazole fbiB SMOR Mtb_fbiBR260-UT2 ACGCACTTGACTTGTCTTCGCCAACA 92 CGCGCACTGCCTCATCCT Nitroimidazole fbiB SMOR Mtb_fbiBF1237-UT1 ACCCAACTGAATGGAGCCTGACCTG 93 GTCCGCGACGAGCTGG Nitroimidazole fbiB SMOR Mtb_fbiBR+87-UT2 ACGCACTTGACTTGTCTTCCGGTGAA 94 TTGATCCGTCGGGAGGTTGA Nitroimidazole fbiB SMOR Mtb_fbiBF217-UT1 ACCCAACTGAATGGAGCTGCGCCGC 95 AAGCTGATCGAGGATG Nitroimidazole fbiB SMOR Mtb_fbiBR464-UT2 ACGCACTTGACTTGTCTTCGCGCGTC 96 CCATGGTGTCGGTGATG Nitroimidazole fbiB SMOR Mtb_fbiBF356-UT1 ACCCAACTGAATGGAGCGCTGCTGC 97 CGGTCGATCCTGACG Nitroimidazole fbiB SMOR Mtb_fbiBR582-UT2 ACGCACTTGACTTGTCTTCGACTGCG 98 ACCTCGGTGACYACCAACTC Nitroimidazole fbiB SMOR Mtb_fbiBF531-UT1 ACCCAACTGAATGGAGCGGTGTCCG 99 CGACCCATACGGCAATGAG Nitroimidazole fbiB SMOR Mtb_fbiBR794-UT2 ACGCACTTGACTTGTCTTCCGGCGAA 100 CGGACCTGCGCAACAGTT Nitroimidazole fbiB SMOR Mtb_fbiBF 701-UT1 ACCCAACTGAATGGAGCGCCGGGCG 101 CCAACGACCTGTTC Nitroimidazole fbiB SMOR Mtb_fbiBR973-UT2 ACGCACTTGACTTGTCTTCCGTCACT 102 GGTGAGATCAGACCGCCACTTG Nitroimidazole fbiB SMOR Mtb_fbiBF874-UT1 ACCCAACTGAATGGAGCCCCGGCCG 103 ACCCGATTCGTGTG Nitroimidazole fbiB SMOR Mtb_fbiBR1093-UT2 ACGCACTTGACTTGTCTTCGGGCGGC 104 ATCGGGGTAGCTGTGTG Nitroimidazole fbiB SMOR Mtb_fbiBF973-UT1 ACCCAACTGAATGGAGCGCTTGCCC 105 GCCGACGCGATAGAA Nitroimidazole fbiB SMOR Mtb_fbiBR1227-UT2 ACGCACTTGACTTGTCTTCGCGGACC 106 AGGTCAGCGGCAAAGATC Nitroimidazole fgd1 SMOR Mtb_fgd1F-38-UT1 ACCCAACTGAATGGAGCGGCGGGTC 107 GCGTTTATGGCATAGGAGTAGAA Nitroimidazole fgd1 SMOR Mtb_fgd1R257-UT2 ACGCACTTGACTTGTCTTCATGACGG 108 CGGGGTTGTAGCGGAAGG Nitroimidazole fgd1 SMOR Mtb_fgd1F148-UT1 ACCCAACTGAATGGAGCATGCCCCG 109 TTCTCGCTGTCCTGGATG Nitroimidazole fgd1 SMOR Mtb_fgd1R465-UT2 ACGCACTTGACTTGTCTTCGTAATAG 110 TCGCCGTCAAAGTCGACGCGGTC Nitroimidazole fgd1 SMOR Mtb_fgd1F386-UT1 ACCCAACTGAATGGAGCCGCCCGGC 111 TGCGTGAATCGGT Nitroimidazole fgd1 SMOR Mtb_fgd1R629-UT2 ACGCACTTGACTTGTCTTCGCCGGCA 112 TCAGCTTCTCGGTGTAGAGC Nitroimidazole fgd1 SMOR Mtb_fgd1F576-UT1 ACCCAACTGAATGGAGCATCTGTAC 113 GTCCGGCAAGGGCGAGGAG Nitroimidazole fgd1 SMOR Mtb_fgd1R795-UT2 ACGCACTTGACTTGTCTTCCGGGTCG 114 TCGATGCTGTGCTTCTGCT Nitroimidazole fgd1 SMOR Mtb_fgd1F705-UT1 ACCCAACTGAATGGAGCGACCCCGA 115 CCCGGAGCTGGCATT Nitroimidazole fgd1 SMOR Mtb_fgd1R953-UT2 ACGCACTTGACTTGTCTTCCGCTGGT 116 CATGTCCTGGTGCGTGAAATAC Nitroimidazole fgd1 SMOR Mtb_fgd1F895-UT1 ACCCAACTGAATGGAGCAATACGTG 117 ACATGGGGCCTGAACCACCTG Nitroimidazole fgd1 SMOR Mtb_fgd1R+63-UT2 ACGCACTTGACTTGTCTTCCCCAGTG 118 CAATCGTCGACTTACCCGTCTG Nitroimidazole fgd2 SMOR Mtb_fgd2F-98-UT1 ACCCAACTGAATGGAGCCTGCGGGG 119 TCATCTCGCCAGGCTAAC Nitroimidazole fgd2 SMOR Mtb_fgd2R167-UT2 ACGCACTTGACTTGTCTTCTCGGTGC 120 GGAATTGTTCGTGGGATAAGAC Nitroimidazole fgd2 SMOR Mtb_fgd2F121-UT1 ACCCAACTGAATGGAGCGCCGCGGG 121 GTGGGTGTCGTCTTATC Nitroimidazole fgd2 SMOR Mtb_fgd2R294-UT2 ACGCACTTGACTTGTCTTCCAACGCC 122 AGGGTCAGCCAGGGAAACAT Nitroimidazole fgd2 SMOR Mtb_fgd2F224-UT1 ACCCAACTGAATGGAGCCAGCGACC 123 ACCTACAGCCATGGCAAGAC Nitroimidazole fgd2 SMOR Mtb_fgd2R389-UT2 ACGCACTTGACTTGTCTTCGAGGCAA 124 ACGCCTGAGCGACGGTG Nitroimidazole fgd2 SMOR Mtb_fgd2F357-UT1 ACCCAACTGAATGGAGCC ATCCGGC 125 CACCGTCGCTCAGG Nitroimidazole fgd2 SMOR Mtb_fgd2R555-UT2 ACGCACTTGACTTGTCTTCCGAGATC 126 CGCTCACCGCTCCACAGC Nitroimidazole fgd2 SMOR Mtb_fgd2F659-UT1 ACCCAACTGAATGGAGCGGCCGGCC 127 GATACGGTGATGGTTG Nitroimidazole fgd2 SMOR Mtb_fgd2R942-UT2 ACGCACTTGACTTGTCTTCGACCGCC 128 CAATTGGCCAGCACTTTCT Nitroimidazole fgd2 SMOR Mtb_fgd2F766-UT1 ACCCAACTGAATGGAGCCCACCCTG 129 GGTAAGCGGGCCGAACT Nitroimidazole fgd2 SMOR Mtb_fgd2R1061-UT2 ACGCACTTGACTTGTCTTCTTGGTGC 130 GGTAGAAGTCGATGGCGGTGAT Nitroimidazole fgd2 SMOR Mtb_fgd2F939-UT1 ACCCAACTGAATGGAGCGT CGGTAC 131 CGATCCCGGCGTCCAC Nitroimidazole fgd2 SMOR Mtb_fgd2R+94-UT2 ACGCACTTGACTTGTCTTCTCGAAGT 132 CCCACACCGTCGGCAACC Nitroimidazole ddn SMOR Mtb-ddnF-16-UT1 ACCCAACTGAAT GGAGCCGCTAGGA 133 TCAGCGTCATGCCGAAATCAC Nitroimidazole ddn SMOR Mtb-ddnR213-UT2 ACGCACTTGACTTGTCTTCCCCACCG 134 TCGCGCAGGAAGTAGAGC Nitroimidazole ddn SMOR Mtb-ddnF-99-UT1 ACCCAACTGAATGGAGCAGGGCACC 135 GTGCGGCGTGACTG Nitroimidazole ddn SMOR Mtb-ddnR147-UT2 ACGCACTTGACTTGTCTTCCAGCAGC 136 GCGACCGGAATCTTCTGG Nitroimidazole ddn SMOR Mtb-ddnF202-UT1 ACCCAACTGAATGGAGCGCGACGGT 137 GGGCGGGTCATTGTC Nitroimidazole ddn SMOR Mtb-ddnR+18-UT2 ACGCACTTGACTTGTCTTCCGGCGAA 138 GTTGGGAACGGTCAGGGTT Nitroimidazole fbiC SMOR Mtb-fbiCF-44-UT1 ACCCAACTGAATGGAGCTGGGGCGT 139 GCGGRTGATATCAGATTGC Nitroimidazole fbiC SMOR Mtb-fbiCR147-UT2 ACGCACTTGACTTGTCTTCGGTCATC 140 GCTATGGCCGCCTCATCCA Nitroimidazole fbiC SMOR Mtb-fbiCF1106-UT1 ACCCAACTGAATGGAGCGCCCTGGC 141 CCGCTTTGGACGA Nitroimidazole fbiC SMOR Mtb-fbiCR1382-UT2 ACGCACTTGACTTGTCTTCGCGGTAT 142 TGCGGCCCTGAGTGTCG Nitroimidazole fbiC SMOR Mtb-fbiCF1345-UT1 ACCCAACTGAATGGAGCTGGGCGCA 143 GCGATCGACACTCAGG Nitroimidazole fbiC SMOR Mtb-fbiCR1618-UT2 ACGCACTTGACTTGTCTTCCGACATC 144 GCGGCGCAACGAATCAG Nitroimidazole fbiC SMOR Mtb-fbiCF1459-UT1 ACCCAACTGAATGGAGCCTCCGGAA 145 CGCATTGACACCGATGTGCT Nitroimidazole fbiC SMOR Mtb-fbiCR1755-UT2 ACGCACTTGACTTGTCTTCGGCGACC 146 TCTCCGACCGACAGCGAGTAG Nitroimidazole fbiC SMOR Mtb-fbiCF1714-UT1 ACCCAACTGAATGGAGCGTGACGCC 147 GACGCCTACTCGCTGTC Nitroimidazole fbiC SMOR Mtb-fbiCR1870-UT2 ACGCACTTGACTTGTCTTCGCGCCTT 148 GACGGCACGAACCAGAT Nitroimidazole fbiC SMOR Mtb-fbiCF1838-UT1 ACCCAACTGAATGGAGCCTACGCCG 149 ATCTGGTTCGTGCCGTCAAG Nitroimidazole fbiC SMOR Mtb-fbiCR2036-UT2 ACGCACTTGACTTGTCTTCACCCAGC 150 GAACCTCGTCGTCCAGGATTTC Nitroimidazole fbiC SMOR Mtb-fbiCF1965-UT1 ACCCAACTGAATGGAGCCTGCGCGA 151 GGCCGGGCTGGAT Nitroimidazole fbiC SMOR Mtb-fbiCR2229-UT2 ACGCACTTGACTTGTCTTCCGGCAAC 152 GGGACGAACTCGGTGAA Nitroimidazole fbiC SMOR Mtb-fbiCF2162-UT1 ACCCAACTGAATGGAGCCCATCTTAA 153 CGTGCTGCGCGATATTCAGGA Nitroimidazole fbiC SMOR Mtb-fbiCR2415-UT2 ACGCACTTGACTTGTCTTCGTTGGCG 154 CCACCTTCGAGCATCACC Nitroimidazole fbiC SMOR Mtb-fbiCF2320-UT1 ACCCAACTGAATGGAGCGGATCATG 155 TTGCACGGCCGCATCTC Nitroimidazole fbiC SMOR Mtb-fbiCR2567-UT2 ACGCACTTGACTTGTCTTCGCCGCAA 156 GCAGGGCGTATGTGGTAGT Nitroimidazole fbiC SMOR Mtb-fbiCF262-UT1 ACCCAACTGAATGGAGCGCAAGGTG 157 TTTATCCCGGTCACCCGGTTAT Nitroimidazole fbiC SMOR Mtb-fbiCR502-UT2 ACGCACTTGACTTGTCTTCCATAGCC 158 CCGTTCGCCGAGCCATTC Nitroimidazole fbiC SMOR Mtb-fbiCF400-UT1 ACCCAACTGAATGGAGCGAGGTGCC 159 GAATTCGGTTGCAAGGAAG Nitroimidazole fbiC SMOR Mtb-fbiCR632-UT2 ACGCACTTGACTTGTCTTCATCGACG 160 GCGCCACCGGTTTGAG Nitroimidazole fbiC SMOR Mtb-fbiCF554-UT1 ACCCAACTGAATGGAGCCGGGCTGT 161 TGCCGCACCTGAACC Nitroimidazole fbiC SMOR Mtb-fbiCR798-UT2 ACGCACTTGACTTGTCTTCCGTCTCG 162 CCGATGCCGACCAACAGA Nitroimidazole fbiC SMOR Mtb-fbiCF741-UT1 ACCCAACTGAATGGAGCGCCGGCCG 163 GTTGTCCATTCCGTT Nitroimidazole fbiC SMOR Mtb-fbiCR1031-UT2 ACGCACTTGACTTGTCTTCCGGCATT 164 CGTCGCCAGACACCAGGTT Nitroimidazole fbiC SMOR Mtb-fbiCF94-UT1 ACCCAACTGAATGGAGCGGGCCCGA 165 GATGGTGTCACGCTGAAC Nitroimidazole fbiC SMOR Mtb-fbiCR342-UT2 ACGCACTTGACTTGTCTTCTAGCTTG 166 CCCGGCACGGTGACGAAC Nitroimidazole fbiC SMOR Mtb-fbiCF996-UT1 ACCCAACTGAATGGAGCGCGCCGCC 167 GAACCTGGTGTCTG Nitroimidazole fbiC SMOR Mtb-fbiCR1207-UT2 ACGCACTTGACTTGTCTTCCGCCCGC 168 CTGTACGTATTTGGGTTGC SMOR: Single Molecule with Overlapping Reads Assay; RDST: Rapid Drug Susceptibility Testing Assay; primer names containing “gene name_F” are forward primers, while primer names containing “gene name_R” are reverse primers.

TABLE 5 Sizes and positions in the related genes of the amplicons produced with specific primers. Expected PCR Designed Product PCR Size with End Forward Reverse Size Universal Begin Posi- Primer Primer (bp) Tails (bp) Position tion pepQf-60 pepQr152 211 247 −60 151 pepQf74 pepQr324 250 286 75 324 pepQf199 pepQr421 222 258 200 421 pepQf351 pepQr535 184 220 352 535 pepQf496 pepQr713 217 253 497 713 pepQf640 pepQr968 327 363 642 968 pepQf899 pepQr + 19 239 275 900 1138 (+19) pepQ_UT_F1 pepQ_UT_R1 519 555 −114 405 pepQ_UT_F2 pepQ_UT_R2 578 614 690 1267 (+148) Rv0678f-57 Rv0678r124 181 217 −57 124 Rv0678f64 Rv0678r255 191 227 65 255 Rv0678f115 Rv0678r336 221 257 116 336 Rv0678f291-1 Rv0678r + 24 231 267 292 522 (+24) RV0678_F1 RV0678_R1 514 550 −6 507 (+9) atpEf-84 atpEr222 306 342 −84 222 atpEf130 atpEr + 96 212 248 131 342 (+96) atpE_F1 atpE_R1 362 398 −91 271 (+25) Mtb_fbiAF-70 Mtb_fbiAR213 283 319 −70 213 Mtb_fbiAF80 Mtb_fbiAR372 292 328 80 372 Mtb_fbiAF313 Mtb_fbiAR600 287 323 313 600 Mtb_fbiAF437 Mtb_fbiAR665 228 264 437 665 Mtb_fbiAF630 Mtb_fbiAR895 265 301 630 895 Mtb_fbiAF839 Mtb_fbiAR + 170 206 839 1009 13 (+13) Mtb_fbiBF-10 Mtb_fbiBR260 270 306 −10 260 Mtb_fbiBF217 Mtb_fbiBR464 247 283 217 464 Mtb_fbiBF356 Mtb_fbiBR582 226 262 356 582 Mtb_fbiBF531 Mtb_fbiBR794 263 299 531 794 Mtb_fbiBF701 Mtb_fbiBR973 272 308 701 973 Mtb_fbiBF874 Mtb_fbiBR1093 219 255 874 1093 Mtb_fbiBF973 Mtb_fbiBR1227 254 290 973 1227 Mtb_fbiBF1237 Mtb_fbiBR + 221 257 1237 1434 87 (+87) Mtb_fgd1F-38 Mtb_fgd1R257 295 331 −38 257 Mtb_fgd1F148 Mtb_fgd1R465 317 353 148 465 Mtb_fgd1F386 Mtb_fgd1R629 243 279 386 629 Mtb_fgd1F576 Mtb_fgd1R795 219 255 576 795 Mtb_fgd1F705 Mtb_fgd1R953 248 284 705 953 Mtb_fgd1F895 Mtb_fgd1R + 179 215 895 1074 63 (+63) Mtb_fgd2F-98 Mtb__fgd2R167 265 301 −98 167 Mtb_fgd2F121 Mtb__fgd2R294 173 209 121 294 Mtb_fgd2F224 Mtb__fgd2R389 165 201 224 389 Mtb_fgd2F357 Mtb__fgd2R555 197 233 357 555 Mtb_fgd2F659 Mtb__fgd2R942 283 319 659 942 Mtb_fgd2F766 Mtb_fgd2R1061 295 331 766 1061 Mtb_fgd2F939 Mtb__fgd2R + 238 274 939 1177 94 (+94) Mtb-ddnF-16 Mtb-ddnR213 229 265 −16 213 Mtb-ddnF-99 Mtb-ddnR147 246 282 −99 147 Mtb-ddnF202 Mtb-ddnR + 272 308 202 474 18 (+18) Mtb-fbiCF-44 Mtb-fbi_CR147 191 227 −44 147 Mtb-fbiCF94 Mtb-fbi_CR342 248 284 94 342 Mtb-fbiCF262 Mtb-fbi_CR502 240 276 262 502 Mtb-fbiCF400 Mtb-fbi_CR632 232 268 400 632 Mtb-fbiCF554 Mtb-fbiCR798 244 280 554 798 Mtb-fbiCF741 Mtb-fbiCR1031 290 326 741 1031 Mtb-fbiCF996 Mtb-fbiCR1207 211 247 996 1207 Mtb-fbiCF1106 Mtb-fbiCR1382 276 312 1106 1382 Mtb-fbiCF1345 Mtb-fbiCR1618 273 309 1345 1618 Mtb-fbiCF1459 Mtb-fbiCR1755 296 332 1459 1755 Mtb-fbiCF1714 Mtb-fbiCR1870 156 192 1714 1870 Mtb-fbiCF1838 Mtb-fbiCR2036 198 234 1838 2036 Mtb-fbiCF1965 Mtb-fbiCR2229 264 300 1965 2229 Mtb-fbiCF2162 Mtb-fbiCR2415 253 289 2162 2415 Mtb-fbiCF2320 Mtb-fbiCR2567 247 283 2320 2567 ddnF ddnR 496 532 −21 475 (19) fbiAF1 fbiAR1 514 550 −25 489 FbiA_F1 FbiA_R1 514 550 −25 489 FbiA_F2 FbiA_R2 513 549 447 959 fbiBF1 fbiBR1 534 570 −73 461 FbiB_F1 FbiB_R1 534 570 −73 461 FbiB_F2 FbiB_R2 426 462 413 838 fbiBF3 fbiBR3 386 422 770 1155 FbiB_F3 FbiB_R3 387 423 769 1155 FbiB_F4 FbiB_R4 305 341 1070 1374 FbiC_F1 FbiC_R1 514 550 −19 495 FbiC_F2 FbiC_R2 498 534 412 909 FbiC_F3 FbiC_R3 514 550 869 1382 FbiC_F4 FbiC_R4 496 532 1343 1838 FbiC_F5 FbiC_R5 391 427 1781 2171 FbiC_F6 FbiC_R6_redo 473 509 2098 2570 Fgd1_F1 Fgd1_R1 485 521 −31 454 Fgd1_F2 Fgd1_R2 504 540 292 795 Fgd1_F3 Fgd1_R3 329 365 753 1081 (+70) Fgd2_F1 Fgd2_R1 450 486 654 1103 (+20) Fgd2_F2 Fgd2_R2 438 474 259 696 Fgd2_F3 Fgd2_R3 351 387 −52 299

TABLE 6 Nucleotide Sequences and Amino Acid Sequences Information of Genes Associated with Drug Resistance Gene Name Protein NCBI NCBI Ref. Position with Respect to NCBI Ref. Ref. Rv0678 Position 778990 to 779487 of NP_215192.1 NC_000962.3 pepQ Position 2859300 to 2860418 of NP_217051.1 NC_000962.3 atpE Position 1461045 to 1461291 of NP_215821.1 NC_000962.3 ddn Position 3987023 to 3987478 of WP_003419309.1 NC_018143.2 fbiA Position 3640543 to 3641538 of NP_217778.1 NC_000962.3 fbiB Position 3641535 to 3642881 of NP_217779.1 NC_000962.3 fbiC Position 1302931 to 1305501 of NP_215689.1 NC_000962.3 fgd Position 490786 to 491796 of WP_003898438.1 NC_018143.2

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We claim:
 1. A method of detecting and/or quantifying a drug-resistant subpopulation of Mycobacterium tuberculosis in a sample, comprising: obtaining an amplicon from the sample, wherein the amplicon comprises a region of interest in Rv0678, pepQ, atpE, ddn, fbiA, fbiB, fbiC, fgd, fgd1, fgd2, or a combination thereof, and the region of interest comprises a polymorphism associated with the drug-resistant subpopulation; sequencing the amplicon; and detecting and/or quantifying a minor variant of the polymorphism in the amplicon, wherein the presence of the minor variant indicates the presence of the drug-resistant subpopulation.
 2. The method of claim 1, wherein the drug-resistant subpopulation of Mycobacterium tuberculosis is resistant to a bedaquiline-related quinolone derivative, a nitroimidazole antibiotic, or both.
 3. The method of claim 1, wherein obtaining the amplicon comprises generating the amplicon using at least one primer comprising a sequence with at least 85% identity to a sequence set forth in SEQ ID NOs: 1-168 or a complement thereof.
 4. The method of claim 1, wherein the minor variant is selected from the group consisting of: a single nucleotide polymorphism (SNP), an insertion, a deletion, and combinations thereof.
 5. The method of claim 4, wherein the minor variant comprises an insertion or deletion in Rv0678 at position 132, 136, 137, 138, 139, 192, 193, or a combination thereof.
 6. The method of claim 5, wherein the minor variant comprises an insertion of G or GA at position 138, an insertion of T at position 139, an insertion of G at position 192, a deletion of G at position 193, or a combination thereof.
 7. The method of 4, wherein the minor variant comprises a SNP in atpE at position 201, 223, or a combination thereof.
 8. The method of claim 7, wherein minor variant comprises a SNP at position 201 where C is replaced with A or G, a SNP at position 223 where G is replaced with C or T, or a combination thereof.
 9. The method of claim 3, wherein the region of interest comprises a polymorphism in Rv0678 associated with the bedaquiline-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 20-30 and a complement thereof.
 10. The method of claim 3, wherein the region of interest comprises a polymorphism in pepQ associated with the bedaquiline-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 1-19 and a complement thereof.
 11. The method of claim 3, wherein the region of interest comprises a polymorphism in atpE associated with the bedaquiline-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 31-34 and a complement thereof.
 12. The method of claim 3, wherein the region of interest comprises a polymorphism in ddn associated with the nitroimidazole-resistant subpopulation, and at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 35-36, 133-138, and a complement thereof.
 13. The method of claim 3, wherein the region of interest comprises a polymorphism in fbiA associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 37-38, 59-60, 77-90, and a complement thereof.
 14. The method of claim 3, wherein the region of interest comprises a polymorphism in fbiB associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 39-42, 55-58, 73-76, 91-106, and a complement thereof.
 15. The method of claim 3, wherein the region of interest comprises a polymorphism in fbiC associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 49-54, 67-72, 139-168, and a complement thereof.
 16. The method of claim 3, wherein the region of interest comprises a polymorphism in fgd associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 43-48, 61-66, and a complement thereof.
 17. The method of claim 3, wherein the region of interest comprises a polymorphism in fgd1 associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 107-118 and a complement thereof.
 18. The method of claim 3, wherein the region of interest comprises a polymorphism in fgd2 associated with the nitroimidazole-resistant subpopulation, and the at least one primer comprises a sequence selected from the group consisting of SEQ ID NOs: 119-132 and a complement thereof.
 19. The method of claim 3, further comprising aligning the sequencing data using an alignment algorithm and interrogating the aligned sequencing data to detect and/or quantify the minor variant of the polymorphism.
 20. The method of claim 1, wherein the steps of sequencing the amplicon and detecting and/or quantifying a minor variant of the polymorphism in the amplicon comprise sequencing two complementary strands of each amplicon to obtain independent sequencing reads of the minor variant and calling the minor variant only when the independent sequencing reads of the minor variant are identical.
 21. The method of claim 1, wherein the sample is selected from the group consisting of: sputum, pleural fluid, blood, saliva, and combinations thereof from a subject.
 22. The method of claim 1, further comprising predicting phenotypic M. tuberculosis resistance to bedaquiline, nitroimidazole, or both, based on a micro-heteroresistance threshold.
 23. The method of claim 22, wherein the micro-heteroresistance threshold is about 5.0%.
 24. The method of claim 1, further comprising administering to the subject a therapeutic agent customized based on the drug resistance of the M. tuberculosis subpopulation in the sample.
 25. The method of claim 24, wherein the therapeutic agent is selected from the group consisting of: an antibiotic, PA-824, OPC-67683, SQ109, TMC207, NAS-21, NAS-91, and combinations thereof.
 26. A primer for detecting and/or quantifying a drug-resistant subpopulation of Mycobacterium tuberculosis in a sample, the primer comprising a sequence with at least 85% identity to a sequence set forth in SEQ ID NOs: 1-168 or a complement thereof; and a label or a modified nucleotide; wherein the primer is between 10 to 70 nucleotides in length.
 27. The primer of claim 26, wherein the sequence of the primer consists of a sequence set forth in SEQ ID NOs: 1-168 or a complement thereof; and a label or a modified nucleotide.
 28. A kit for detecting and/or quantifying a drug-resistant subpopulation of Mycobacterium tuberculosis in a sample, comprising a primer of claim 26 and reagents for amplification of a genomic sample. 